Pyrosequencing
Mostaga Ronaghi and Pal Nyren developed pyrosequencing at the Royal Institute of Technology in Stockholm in 1996 1. Pyrosequencing is a method used to determine the order of nucleotides in DNA, or DNA sequencing, that is based on the detection of released pyrophosphate (PPi) during DNA synthesis, and it is different from Sanger sequencing because it does not rely on chain termination with dideoxynucleotides 1,2. Standard pyrosequencing uses the Klenow fragment of Escherichia coli DNA Polymerase I. This is a relatively slow polymerase. Also, a recombinant form of ATP sulfurylase, which is from the yeast Saccharomyces cerevisiae, as well as the luciferase from the American firefly Photius pyralis is used. The overall reaction from polymerization to light detection takes places within a few seconds at room temperature. Complimentary nucleotides are incorporated into the single strand DNA templates. During a cascade of these enzymatic reactions, visible light is generated. The light that is emitted is proportional to the number of nucleotides that are incorporated as well as if there are more than one of each nucleotide in a row. The amount of light that is being emitted can be detected via a photodiode, photomultiplier tube, or a charge-coupled device (CCD) camera 3. There are two types of pyrosequencing that are conmmonly used. The first is solid-phase pyrosequencing, which uses immobilized DNA in a three-enzyme system. This method includes a washing step in order to remove the excess substrate after the addition of each nucleotide. The second is liquid-phase pyrosequencing, where apyyrase, a nucleotide-degrading enzyme from potato is used to make a four-enzyme system to eliminate the need for solid support as well as the intermediate washing. This allows the pyrosequencing reaction to be performed in a single tube 3. First, the DNA that is being sequenced is broken up into fragments of about 100 base pairs and denatured to form ssDNA. The ssDNA fragments are attached to microscopic beads, which are separated from other. A polymerase chain reaction (PCR) is run on each bead in order to coat each bead with about 10 million identical copies of that specific fragment. The beads are placed into separate microscopic wells, and they receive the following reagents: #DNA polymerase for adding deoxynucleotides to the ssDNA #Adenosine phosphosulfate (APS) # ATP sulfurylase to form ATP from adenosine phosphosulfate (APS) and pyrophosphate (PPi) #Luciferin # Luciferase to catalyze the conversion of luciferin into oxyluciferin and generate visible light The cascade of enzymatic reactions begins with a nucleic acid polymerization reaction. Incorporation of new nucleotides results in the release of inorganic PPi by polymerase. ATP sulfurylase converts the released PPi into ATP, and luciferase uses this ATP to oxidize luciferin and generate light. The sequence of the template can be determined easily, because the added nucleotide is known. The previous nucleotide letter is always degraded before the next letter is added for synthesis so that the next complementary letter in the sequence can be revealed. This process is repeated with each of the four letters until the DNA sequence of the single stranded template is determined 1. References: 1) http://en.wikipedia.org/wiki/Pyrosequencing 2) http://genome.cshlp.org/content/11/1/3.full 3) http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/P/Pyrosequencing.html 4) http://bitesizebio.com/19008/how-bisulfite-pyrosequencing-works/